1. Field of the Invention
The present invention relates to biomolecules which are bound to catalytic inorganic particles, immunoassays which utilize such biomolecules, and kits for carrying out such immunoassays.
2. Discussion of the Background
The detection of trace amounts of biologically significant compounds, such as steroids or drugs of abuse, is often accomplished quickly and inexpensively by the employment of an immunoassay. Such an assay relies on an immunogenic recognition of the substance in question followed by the amplification of that recognition. Enzymes are widely used in immunoassays as the amplifier of the antibody-antigen recognition event. One of the most common types of immunoassays is the Enzyme-Linked Immunosorbant Assay (ELISA).
ELISA may be preformed in a number of different ways. The two most common are the competitive mode and the sandwich assay. In a competitive mode ELISA, a surface, usually either a polystyrene plate or a nitrocellulose membrane, is coated with a capture antigen. These surfaces are normally chosen because they bind protein non-specifically. Therefore, if the antigen is not a protein, it may be covalently linked to a carrier protein and bound to the surface without further chemistry. After the antigen is bound, the remaining binding sites on the surface are blocked with another protein. Then the test fluid and enzyme-labeled antibody are added. If no antigen is in the test fluid, all the labeled antibody will bind to the antigen adsorbed on the surface. Conversely, if antigen is present in the test fluid, the antigen will block the binding sites on the enzyme-labeled antibody and prevent it from binding to the antigen adsorbed on the surface. The surface is washed to remove unbound materials, and a substrate is added for the enzyme. The enzyme catalyzes a reaction in which the substrate reacts to form a colored material which can be quantitatively observed with a spectrophotometer. The intensity of the color produced is proportional to the enzyme activity and the amount of antibody bound, which is inversely proportional to the amount of antigen in the test fluid.
In a sandwich assay ELISA, an antibody that recognizes part of the antigen is bound to a surface. Since antibodies are proteins, this is readily accomplished by allowing the surface to contact a solution of the antibody. As in the competitive ELISA, the remaining sites on the surface are blocked with another protein. The test fluid is then added. If an antigen is present in the test fluid, the antibody on the surface will capture the antigen. Then a second, enzyme-labeled antibody, which recognizes a different part of the antigen than the first antibody, is added. The second antibody will then bind to the antigen which is captured on the surface. After washing the surface to remove any unbound materials, a substrate for the enzyme is added and the color produced is observed spectrophotometrically. In this form of an ELISA, the signal is directly proportional to the concentration of the antigen in a test sample. Such a sandwich assay is widely used in the commercial arena for home pregnancy tests.
In either type of ELISA, the enzyme acts as the amplifier of the antigen-antibody reaction. That is, a color or other signal, such as light from some chemiluminescent reaction, is produced that can be observed macroscopically. Without this amplification step, the sensitivity of an immunoassay would be orders of magnitude less.
Several problems occur in the use of enzymes as amplifiers in immunoassays. They are:
1. Any change in enzyme activity will affect the precision of the assay. For example, loss of half of the activity of the enzyme in a competitive ELISA may produce a false positive since less signal indicates the presence of the test substance. Since enzyme activity is sensitive to storage conditions, enzymes must be kept either refrigerated, freeze dried or both. Also, controls must be performed to constantly test the activity of the enzyme. Inevitably, the shelf-life is limited by the stability of the enzyme.
2. Enzymes are expensive. Being derived from living sources, they require substantial processing costs. The least expensive enzyme, on an activity basis, is Horseradish Peroxidase which is not surprisingly the most common enzyme used in ELISAs. However, even Horseradish Peroxidase costs about $5/mg or $5000/g, a cost which is about 450 times the cost of gold. Fortunately, very little enzyme is necessary for each assay.
3. The labeling of antibodies with enzymes is often a quite laborious procedure as one must ensure that little unbound enzyme is present. If significant amounts of unbound enzyme are present or significant amounts of unlabeled antibody are present, the sensitivity of the ELISA is reduced.
4. Enzymes are often heterogeneous materials due to their isolation from natural sources. Therefore, characterization of enzyme-antibody conjugates can be difficult.
Enzymes are also used for detection of the hybridization of DNA or RNA to its complimentary strand, often in conjunction with amplification of the DNA or RNA target by the polymerase chain reaction (PCR). These reactions are widely employed for DNA fingerprinting, and the detection of genetic defects, viruses and bacteria. Because the PCR reaction requires heating and cooling of the reaction mixture to cause denaturation of the DNA, the common enzymes such as peroxidase or alkaline phosphatase cannot be added to the reaction mixture until after the amplification reactions occur. This limits some of the procedures that can be preformed. Catalytic particles do not have this limitation and therefore give more flexibility to the detection of nucleic acids.
Colloidal metals have been employed in immunoassays previously. Mostly, they consisted of either colloidal iron or gold (M. Horisberger, "Colloidal Gold: A Cytochemical Marker for Light and Fluorescent Microscopy and for Transmission and Scanning Electron Microscopy", Scanning Electron Microscopy, pp. 19-40 (1981); and Martin et al, "Characterization of Antibody Labelled Colloidal Gold Particles and Their Applicability in a sol Particle Immunoassay, SPIA", J. Immunoassay, vol. 11, pp. 31-48 (1990)). However, in either case, the metals were only chosen for their color, i.e., their presence is determined only by their color or electron density under an electron microscope. Both the color and electron density are directly proportional to the mass of the metal colloid, not their catalytic activity. Thus a relatively large amount of material is necessary to be observed and they can only compete in sensitivity to enzyme-type amplifiers of the antibody-antigen reaction when the signal is further amplified by an instrument such as an electron microscope.
Similarly, the use of colloidal gold and colloidal silver as markers in histochemistry has also been reported. (Lucocq and Roth, "Colloidal Gold and Colloidal Silver-Metallic Markers for Light Microscopic Histochemistry", Techniques in Immunochemistry, vol. 3, pp. 203-236 (1985)). Again, the colloidal particles were not detected on the basis of any catalytic activity. More recently amplification of gold colloids has occurred via a process very similar to photography. The gold colloids act as nucleation sites for the precipitation of silver, which is the colorimetric material (see p. 273 of the 1993 BioRad Life Sciences Research Product Catalog, Hercules, Calif.).
Stable colloidal rhodium (0) suspensions have been reported to catalyze the hydrogenation of liquid alkenes in biphasic systems under mild conditions (Larpent et al, "New Highly Water-Soluble Surfactants Stabilize Colloidal Rhodium (0) suspensions Useful in Biphasic Catalysts", J. Molecular Catalysis, vol. 65, pp. L35-L40 (1991)). However, there is no report of such colloidal rhodium particles being bound to a biomolecule, such as an antigen.
The oxidation of luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) by hydrogen peroxide is a chemiluminescent reaction known to be catalyzed by colloidal platinum (Albrecht, Z. Phys. Chem., vol. 136, p. 321 (1928)). However, there is no report of such colloidal platinum being bound to a biomolecule.
Tris(2,2'-bipyridine)ruthenium II has been used as a peroxide-producing replacement for an enzyme label (Ismail and Weber, "Tris-2,2'-Bipyridineruthenium-II as a Peroxide-Producing Replacement for Enzymes as Chemical Labels", Biosens. Bioelectronics, vol. 6, pp. 698-705 (1991). However, the hydrogen peroxide is produced by photolysis with such compounds, and accordingly, the use of such labels in an assay requires the use of photolysis equipment.